The detection of antibodies in biological samples to help in the diagnosis of diseases, infection or an immune reaction is known in the art. For some indications, such as autoimmune diseases, these antibodies are autoantibodies that recognize and complex with “self antigens,” molecules within a person's own body that are capable of stimulating autoimmunity. Autoimmune disease arises when the body initiates an immune response against its own tissues and organs. When this occurs, the immune system produces antibodies, also known as autoantibodies that target and attack cells or tissues of the body. This reaction is called an autoimmune response and is characterized most commonly by inflammation and tissue damage. Therefore, autoimmune diseases are a major health risk worldwide. The actual number of individuals that suffer from autoimmune disease is not known due to lack of recorded statistics. However, this number is expected to increase dramatically in the next decade because of a number of factors, including increasing environmental pollution. In particular, environmental pollution, such as ultraviolet radiation, ozone, organic solvents and ultrafine particles have been linked to inducing and/or exacerbating autoimmune diseases. Today, there are over eighty illnesses caused by autoimmunity, and several others are believed to be the result of this condition. Approximately 5-7% of all Americans are affected by these diseases and over 75% of those are women. In fact, it is one of the ten leading causes of death in women in all age groups up to 65 years. By comparison, approximately 23.5 million people in the United States suffer from autoimmune disease as compared to 9.0 million that have cancer.
Some autoimmune diseases are tissue- or organ-specific, while others affect several organs of the human body. Diseases of this latter category are often termed “systemic autoimmune rheumaticdiseases (“SARD”), and the symptoms may vary from one patient to the next, with tissue injury and inflammation occurring in multiple sites and organs without relation to their antigenic makeup. Some of the more common SARD include rheumatoid arthritis, systemic lupus erythematosus (lupus), mixed connective tissue disease systemic sclerosis, polymyositis, dermatomyositis, Sjögren's syndrome, and the like. Autoimmune diseases are generally believed to be influenced by multiple factors, with some of the contributing factors being genetic disposition, host factors (such as T cell defects and polyclonal stimulation of B cells that are resistant to controls), environmental factors (such as viruses, chemical agents and certain microbial infections), and antigen-driven mechanisms (such as sequestered antigens or cross-reacting exogenous antigens).
A common characteristic of many SARD is the presence of one or more types of antinuclear antibodies (“ANA”) in the bodily fluids of affected patients. Generally, ANAs are autoantibodies directed against antigens in the nucleus of a person's own cells. Thus, various diagnostic ANA assays and screens have been formulated over the years for the detection of ANAs, such as indirect immunofluorescence (“IIF”) assays and enzyme linked immunosorbent assays (“ELISAs”).
The IIF assay is one of the most commonly used routine tests for the detection of ANAs and is recommended by the American College of Rheumatology (“ACR”). Typically, a patient's serum is diluted in a buffer solution and allowed to react with cells that have been fixed on a glass slide. If there are antibodies in the patient's serum that are immunoreactive with antigen components associated with the cell, they will bind to the cells and form an antigen-antibody complex. After washing to remove any unbound material, the presence of antigen-antibody complexes is detected using an anti-human antibody labeled with a fluorescent moiety. The presence of a fluorescent signal is then detected by viewing the cells under a microscope or more recently with digital imaging systems.
The IIF method is not specific for certain autoimmune diseases because various autoantibody specificities are detected which have different clinical associations. In a significant number of cases, multiple overlapping fluorescent patterns can substantially complicate the interpretation of the pattern. Therefore, specific confirmation tests are mandatory to identify the autoantibody specificities present in the patient's specimen. More specifically, even if a pattern is seen that is suggestive of a specific autoimmune disease, extensive confirmatory testing with purified antigens such as Sm, Sc1-70, Ro, La, RNP and double stranded DNA (“dsDNA”), using assays such as enzyme immunoassays (“EIAs”), immunodiffusion or hemagglutination is necessary before one can utilize the test results to help in the diagnosis of the disease. In addition, obscuring of fluorescent patterns can occur, especially if the sample is not titered appropriately. Recent studies have shown that IIF on human epithelial line (“HEp-2”) cells has a false positive rate of about 20%. Specifically, when ANA diagnostic tests produce a false positive result, clinicians will typically order a series of confirmatory tests that are both costly and can lead to misdiagnoses. Thus, one of the drawbacks to using the IIF method, for ANA detection, is that it generates a high number of false positives, which can lead to misdiagnosis of patients suffering from an autoimmune disease or conditions that mimic an autoimmune disease, and subsequently unnecessary treatment or insufficient treatment.
Additionally, results can be difficult to interpret and as such, the utilization of specific antibody results is typically less than optimal. Moreover, the delay between the generation of a positive screen and the generation of the specific antibody results can be difficult both for the patient and for the clinician, given the low positive predictive value of the initial result. In particular, when specific antibody results are received, they may be highly suggestive in some eases (e.g., positive anti-Sc1-70 antibody, indicating high likelihood of systemic sclerosis) or they may not be very useful (e.g., positive Ro52 alone, clinical association still unknown). As such, a positive ANA test may yield a sizable portion of ANA-positive individuals with no confirming evidence of autoimmune disease. This becomes even more crucial in view of the perception that autoantibodies may precede the clinical onset of autoimmune disease for many years.
Thus, IIF is a diagnostic method with obvious limitations that does not generate a permanent record, involves multiple assays, is time-consuming, labor intensive and expensive (when personnel and follow-up test costs are considered), requires considerable expertise in the interpretation of results and can lead to misdiagnoses of autoimmune disease.
In recent years, approximately 20% of healthy individuals have been reported to contain ANA, in which anti-dense fine speckles 70 (“anti-DFS70”) antibodies represent a major cause of false positive results. Anti-DFS70 antibodies were initially identified as an ANA from a patient with interstitial cystitis, and those autoantibodies were later associated with various other disease conditions such as atopic dermatitis (Ochs et al, 1994 J Urol; 151:587-592). Ochs et al. described the IIF staining pattern by anti-DFS70 antibodies as a characteristic immunohistochemical staining pattern on HEp-2 cells consisting of DFS distributed in the nucleoplasm in interphase cells and with accentuated generalized staining of condensed chromosomes in mitotic cells. Although a 70-kDa protein was recognized by immunoblotting and the antigen was initially termed DFS70, the primary target autoantigen was subsequently identified as the lens epithelium—derived growth factor (LEDGF), also called DNA binding transcription coactivator p75. This protein is believed to have a number of physiological functions, including serving as a cofactor for human immunodeficiency virus replication through an interaction with viral integrase, and it is also highly expressed in prostate tumor tissue.
Since their first characterization, anti-DFS70 antibodies have been found in the sera of patients with a variety of chronic inflammatory conditions, cancer patients, and even frequently in healthy individuals. In 2005, Dellavance et al. (Dellavance et al. 2005 J Rheumatol 32(11):2144-9) evaluated over 10,000 ANA positive samples by HF and then immunoblot, reporting that anti-DFS70 antibodies were common among ANA-positive individuals with no evidence of SARD, and that among autoimmune patients with this autoantibody, over 50% had evidence of autoimmune thyroiditis. The highest prevalence of anti-DFS70 antibodies has been reported in patients with Vogt-Harada syndrome (66.7%) and atopic dermatitis (30%), followed by healthy individuals (˜10%), while its prevalence in SARD is significantly lower (˜2-3%). Furthermore, when considering the prognostic and long term outcome of individuals who have anti-DFS70 antibodies, it was recently reported that none out of 40 anti-DFS70 positive healthy individuals developed autoimmune disease within an average 4-year interval. Therefore, it has been suggested that anti-DFS antibodies may be utilized as a biomarker to rule out SARD.
However, the potential clinical significance of autoantibodies to DFS70/LEDGF has also been questioned due to conflicting studies. In 2004, Watanabe et al. (Watanabe et al. 2004 Arthritis Rheum 50:892-900) showed that symptoms associated with atopic dermatitis were present among anti-DFS70 positive subjects. Further in contrast, Yamada et al. (Yamada et al. 2001 Immunol Lett 78:161-8) used ELISA to show a high prevalence of autoantibodies to DFS70 in patients with Vogt-Harada syndrome, a systemic autoimmune and inflammatory disorder. Other studies have also demonstrated that autoantibodies to DFS70/LEDGF occur in a variety of chronic inflammatory conditions. Thus, the clinical significance of testing for anti-DFS70 antibodies is still a point of debate as to its association and relevance to diagnosing autoimmune disease. However, a consensus was achieved that anti-DFS70 antibodies are significantly less prevalent in patients with SARD compared to healthy individuals. Therefore, anti-DFS70 antibodies reduce the specificity and thus the positive predictive value of the ANA test.
Accordingly, there is a need in the field for a method that increases the specificity of antibody-based tests for autoimmune diseases that reduces the frequency of false positives in ANA detection, high cost, unnecessary confirmatory testing and misdiagnosis of autoimmune diseases.